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Cardiac troponin C (cTnC) is the critical Ca 2+ -sensing component of the troponin complex. Binding of Ca 2+ to cTnC triggers a cascade of conformational changes within the myofilament that culminate in force production. Hypertrophic cardiomyopathy (HCM)-associated TNNC1 variants generally induce a greater degree and duration of Ca 2+ binding, which may underly the hypertrophic phenotype. Regulation of contraction has long been thought to occur exclusively through Ca 2+ binding to site II of cTnC. However, work by several groups including ours suggest that Mg 2+ , which is several orders of magnitude more abundant in the cell than Ca 2+ , may compete for binding to the same cTnC regulatory site. We previously used isothermal titration calorimetry (ITC) to demonstrate that physiological concentrations of Mg 2+ may decrease site II Ca 2+ -binding in both N-terminal and full-length cTnC. Here, we explore the binding of Ca 2+ and Mg 2+ to cTnC harbouring a series of TNNC1 variants thought to be causal in HCM. ITC and thermodynamic integration (TI) simulations show that A8V, L29Q and A31S elevate the affinity for both Ca 2+ and Mg 2+ . Further, L48Q, Q50R and C84Y that are adjacent to the EF hand binding motif of site II have a more significant effect on affinity and the thermodynamics of the binding interaction. To the best of our knowledge, this work is the first to explore the role of Mg 2+ in modifying the Ca 2+ affinity of cTnC mutations linked to HCM. Our results indicate a physiologically significant role for cellular Mg 2+ both at baseline and when elevated on modifying the Ca 2+ binding properties of cTnC and the subsequent conformational changes which precede cardiac contraction., (© 2022 Federation of European Biochemical Societies.)

Several mutations identified in phospholamban (PLN) have been linked to familial dilated cardiomyopathy (DCM) and heart failure, yet the underlying molecular mechanism remains controversial. PLN interacts with sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) and regulates calcium uptake, which is modulated by the protein kinase A (PKA)-dependent phosphorylation of PLN during the fight-or-flight response. Here, we present the crystal structures of the catalytic domain of mouse PKA in complex with wild-type and DCM-mutant PLNs. Our structures, combined with the results from other biophysical and biochemical assays, reveal a common disease mechanism: the mutations in PLN reduce its phosphorylation level by changing its conformation and weakening its interactions with PKA. In addition, we demonstrate that another more ubiquitous SERCA-regulatory peptide, called another-regulin (ALN), shares a similar mechanism mediated by PKA in regulating SERCA activity., Competing Interests: JQ, JZ, LL, OH, ZL, KW, FV, YZ, ZY No competing interests declared, (© 2022, Qin et al.)

Ryanodine Receptors (RyRs) are massive channels that release Ca 2+ from the endoplasmic and sarcoplasmic reticulum. Hundreds of mutations are linked to malignant hyperthermia (MH), myopathies, and arrhythmias. Here, we explore the first MH mutation identified in humans by providing cryo-EM snapshots of the pig homolog, R615C, showing that it affects an interface between three solenoid regions. We also show the impact of apo-calmodulin (apoCaM) and how it can induce opening by bending of the bridging solenoid, mediated by its N-terminal lobe. For R615C RyR1, apoCaM binding abolishes a pathological 'intermediate' conformation, distributing the population to a mixture of open and closed channels, both different from the structure without apoCaM. Comparisons show that the mutation primarily affects the closed state, inducing partial movements linked to channel activation. This shows that disease mutations can cause distinct pathological conformations of the RyR and facilitate channel opening by disrupting interactions between different solenoid regions.

The diamide insecticide class is one of the top-selling insecticides globally. They are used to control a wide range of pests by targeting their ryanodine receptors (RyRs). Here, we report the highest-resolution cryo-electron microscopy (cryo-EM) structure of RyR1 in the open state, in complex with the anthranilic diamide chlorantraniliprole (CHL). The 3.2-Å local resolution map facilitates unambiguous assignment of the CHL binding site. The molecule induces a conformational change by affecting the S4-S5 linker, triggering channel opening. The binding site is further corroborated by mutagenesis data, which reveal how diamide insecticides are selective to the Lepidoptera group of insects over honeybee or mammalian RyRs. Our data reveal that several pests have developed resistance via two mechanisms, steric hindrance and loss of contact. Our results provide a foundation for the development of highly selective pesticides aimed at overcoming resistance and therapeutic molecules to treat human myopathies.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the progressive loss of cortical, brain stem and spinal motor neurons that leads to muscle weakness and death. A previous study implicated CACNA1H encoding for Ca v 3.2 calcium channels as a susceptibility gene in ALS. In the present study, two heterozygous CACNA1H variants were identified by whole genome sequencing in a small cohort of ALS patients. These variants were functionally characterized using patch clamp electrophysiology, biochemistry assays, and molecular modeling. A previously unreported c.454GTAC > G variant produced an inframe deletion of a highly conserved isoleucine residue in Ca v 3.2 (p.ΔI153) and caused a complete loss-of-function of the channel, with an additional dominant-negative effect on the wild-type channel when expressed in trans. In contrast, the c.3629C > T variant caused a missense substitution of a proline with a leucine (p.P1210L) and produced a comparatively mild alteration of Ca v 3.2 channel activity. The newly identified ΔI153 variant is the first to be reported to cause a complete loss of Ca v 3.2 channel function. These findings add to the notion that loss-of-function of Ca v 3.2 channels associated with rare CACNA1H variants may be risk factors in the complex etiology of ALS.

Ryanodine receptors (RyRs) are intracellular Ca 2+ release channels controlling essential cellular functions. RyRs are targeted by cyclic AMP (cAMP)-dependent protein kinase A (PKA), a controversial regulation implicated in disorders ranging from heart failure to Alzheimer's. Using crystal structures, we show that the phosphorylation hotspot domain of RyR2 embraces the PKA catalytic subunit, with an extensive interface not seen in PKA complexes with peptides. We trapped an intermediary open-form PKA bound to the RyR2 domain and an ATP analog, showing that PKA can engage substrates in an open form. Phosphomimetics or prior phosphorylation at nearby sites in RyR2 either enhance or reduce the activity of PKA. Finally, we show that a phosphomimetic at S2813, a well-known target site for calmodulin-dependent kinase II, induces the formation of an alpha helix in the phosphorylation domain, resulting in increased interactions and PKA activity. This shows that the different phosphorylation sites in RyR2 are not independent., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Murine antibodies S25-23, S25-26, and S25-5 derive from a common germ-line origin, and all bind the Chlamydiaceae family-specific epitope αKdo(2→8)αKdo(2→4)αKdo (where Kdo is 3-deoxy-α-d- manno-oct-2-ulosonic acid) with high affinity and specificity. These antibodies recognize the entire trisaccharide antigen in a linkage-dependent manner via a groove composed largely of germ-line residues. Despite sharing identical heavy and light chain genes, S25-23 binds the family-specific epitope with nanomolar affinity, which is an order of magnitude higher than that of S25-26, while S25-5 displays an affinity between those of S25-23 and S25-26. We determined the high-resolution crystal structures of S25-23 and S25-5 antigen binding fragments in complex with a pentasaccharide derived from the LPS of Chlamydia and measured the affinity of S25-5 for chlamydial LPS antigens using isothermal titration microcalorimetry. The 1.75 Å resolution structure of S25-23 shows how subtle conservative mutations Arg(L)-27E to lysine and Ser(H)-56 to threonine lead to an order of magnitude increase in affinity. Importantly, comparison between previous S25-26 structures and the 1.99 and 2.05 Å resolution liganded and unliganded structures of S25-5, respectively, shows how a Ser(L)-27E mutation results in an intermediate affinity due to the reduced enthalpic penalty associated with complex formation that would otherwise be required for arginine in this position. This strategy allows for subtle adjustments in the combining site via affinity maturation that have dramatic consequences for the affinity of an antibody for its antigen.

Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is often a life-threatening arrhythmia disorder with variable penetrance and expressivity. Little is known about the incidence or outcomes of CPVT patients with ≥2 variants., Methods: The phenotypes, genotypes and outcomes of patients in the Pediatric and Congenital Electrophysiology Society CPVT Registry with ≥2 variants in genes linked to CPVT were ascertained. The American College of Medical Genetics & Genomics (ACMG) criteria and structural mapping were used to predict the pathogenicity of variants (3D model of pig RyR2 in open-state)., Results: Among 237 CPVT subjects, 193 (81%) had genetic testing. Fifteen patients (8%) with a median age of 9 years (IQR 5-12) had ≥2 variants. Sudden cardiac arrest occurred in 11 children (73%), although none died during a median follow-up of 4.3 years (IQR 2.5-6.1). Thirteen patients (80%) had at least two RYR2 variants, while the remaining two patients had RYR2 variants plus variants in other CPVT-linked genes. Among all variants identified, re-classification of the commercial laboratory interpretation using ACMG criteria led to the upgrade from variant of unknown significance (VUS) to pathogenic/likely pathogenic (P/LP) for 5 variants, and downgrade from P/LP to VUS for 6 variants. For RYR2 variants, 3D mapping using the RyR2 model suggested that 2 VUS by ACMG criteria were P/LP, while 2 variants were downgraded to likely benign., Conclusions: This severely affected cohort demonstrates that a minority of CPVT cases are related to ≥2 variants, which may have implications on family-based genetic counselling. While multi-variant CPVT patients were at high-risk for sudden cardiac arrest, there are insufficient data to conclude that this genetic phenomenon has prognostic implications at present. Further research is needed to determine the significance and generalizability of this observation. This study also shows that a rigorous approach to variant re-classification using the ACMG criteria and 3D mapping is important in reaching an accurate diagnosis, especially in the multi-variant population., Competing Interests: Drs. Roston & Sanatani are consultants for Audentes Therapeutics. These do not alter our adherence to PLOS ONE policies on sharing data and materials. No other authors have relevant competing interests.

Self-assembling protein surface (S-) layers are common cell envelope structures of prokaryotes and have critical roles from structural maintenance to virulence. S-layers of Gram-positive bacteria are often attached through the interaction of S-layer homology (SLH) domain trimers with peptidoglycan-linked secondary cell wall polymers (SCWPs). Here we present an in-depth characterization of this interaction, with co-crystal structures of the three consecutive SLH domains from the Paenibacillus alvei S-layer protein SpaA with defined SCWP ligands. The most highly conserved SLH domain residue SLH-Gly29 is shown to enable a peptide backbone flip essential for SCWP binding in both biophysical and cellular experiments. Furthermore, we find that a significant domain movement mediates binding by two different sites in the SLH domain trimer, which may allow anchoring readjustment to relieve S-layer strain caused by cell growth and division.

Homologous glycosyltransferases GTA and GTB perform the final step in human ABO(H) blood group A and B antigen synthesis by transferring the sugar moiety from donor UDP-GalNAc/UDP-Gal to the terminal H antigen disaccharide acceptor. Like other GT-A fold family 6 glycosyltransferases, GTA and GTB undergo major conformational changes in two mobile regions, the C-terminal tail and internal loop, to achieve the closed, catalytic state. These changes are known to establish a salt bridge network among conserved active site residues Arg188, Asp211 and Asp302, which move to accommodate a series of discrete donor conformations while promoting loop ordering and formation of the closed enzyme state. However, the individual significance of these residues in linking these processes remains unclear. Here, we report the kinetics and high-resolution structures of GTA/GTB mutants of residues 188 and 302. The structural data support a conserved salt bridge network critical to mobile polypeptide loop organization and stabilization of the catalytically competent donor conformation. Consistent with the X-ray crystal structures, the kinetic data suggest that disruption of this salt bridge network has a destabilizing effect on the transition state, emphasizing the importance of Arg188 and Asp302 in the glycosyltransfer reaction mechanism. The salt bridge network observed in GTA/GTB structures during substrate binding appears to be conserved not only among other Carbohydrate Active EnZyme family 6 glycosyltransferases but also within both retaining and inverting GT-A fold glycosyltransferases. Our findings augment recently published crystal structures, which have identified a correlation between donor substrate conformational changes and mobile loop ordering.

The process of natural selection favours germ-line gene segments that encode CDRs that have the ability to recognize a range of structurally related antigens. This presents an immunological advantage to the host, as it can confer protection against a common pathogen and still cope with new or changing antigens. Cross-reactive and polyspecific antibodies also play a central role in autoimmune responses, and a link has been shown to exist between auto-reactive B cells and certain bacterial infections. Bacterial DNA, lipids, and carbohydrates have been implicated in the progression of autoimmune diseases such as systemic lupus erythematosus. As well, reports of anti-lipid A antibody polyspecificity towards single-stranded DNA together with the observed sequence homology amongst isolated auto- and anti-lipid A antibodies has prompted further study of this phenomenon. Though the lipid A epitope appears cryptic during Gram-negative bacterial infection, there have been several reported instances of lipid A-specific antibodies isolated from human sera, some of which have exhibited polyspecificity for single stranded DNA. In such cases, the breakdown of negative selection through polyspecificity can have the unfortunate consequence of autoimmune disease. This review summarizes current knowledge regarding such antibodies and emphasizes the features of S1-15, A6, and S55-5, anti-lipid A antibodies whose structures were recently determined by X-ray crystallography.

Aberrant glycosylation and the overexpression of specific carbohydrate epitopes is a hallmark of many cancers, and tumor-associated oligosaccharides are actively investigated as targets for immunotherapy and diagnostics. Wisteria floribunda agglutinin (WFA) is a legume lectin that recognizes terminal N-acetylgalactosaminides with high affinity. WFA preferentially binds the disaccharide LacdiNAc (β-d-GalNAc-[1→4]-d-GlcNAc), which is associated with tumor malignancy in leukemia, prostate, pancreatic, ovarian, and liver cancers and has shown promise in cancer glycobiomarker detection. The mechanism of specificity for WFA recognition of LacdiNAc is not fully understood. To address this problem, we have determined affinities and structure of WFA in complex with GalNAc and LacdiNAc. Affinities toward Gal, GalNAc, and LacdiNAc were measured via surface plasmon resonance, yielding K D values of 4.67 × 10 -4 m, 9.24 × 10 -5 m, and 5.45 × 10 -6 m, respectively. Structures of WFA in complex with LacdiNAc and GalNAc have been determined to 1.80-2.32 Å resolution. These high resolution structures revealed a hydrophobic groove complementary to the GalNAc and, to a minor extent, to the back-face of the GlcNAc sugar ring. Remarkably, the contribution of this small hydrophobic surface significantly increases the observed affinity for LacdiNAc over GalNAc. Tandem MS sequencing confirmed the presence of two isolectin forms in commercially available WFA differing only in the identities of two amino acids. Finally, the WFA carbohydrate binding site is similar to a homologous lectin isolated from Vatairea macrocarpa in complex with GalNAc, which, unlike WFA, binds not only αGalNAc but also terminal Ser/Thr O-linked αGalNAc (Tn antigen)., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Lipopolysaccharide dispersed in the blood by Gram-negative bacteria can be a potent inducer of septic shock. One research focus has been based on antibody sequestration of lipid A (the endotoxic principle of LPS); however, none have been successfully developed into a clinical treatment. Comparison of a panel of anti-lipid A antibodies reveals highly specific antibodies produced through distinct germ line precursors. The structures of antigen-binding fragments for two homologous mAbs specific for lipid A, S55-3 and S55-5, have been determined both in complex with lipid A disaccharide backbone and unliganded. These high resolution structures reveal a conserved positively charged pocket formed within the complementarity determining region H2 loops that binds the terminal phosphates of lipid A. Significantly, this motif occurs in unrelated antibodies where it mediates binding to negatively charged moieties through a range of epitopes, including phosphorylated peptides used in diagnostics and therapeutics. S55-3 and S55-5 have combining sites distinct from anti-lipid A antibodies previously described (as a result of their separate germ line origin), which are nevertheless complementary both in shape and charge to the antigen. S55-3 and S55-5 display similar avidity toward lipid A despite possessing a number of different amino acid residues in their combining sites. Binding of lipid A occurs independent of the acyl chains, although the GlcN-O6 attachment point for the core oligosaccharide is buried in the combining site, which explains their inability to recognize LPS. Despite their lack of therapeutic potential, the observed motif may have significant immunological implications as a tool for engineering recombinant antibodies., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Homologous glycosyltransferases α-(1→3)-N-acetylgalactosaminyltransferase (GTA) and α-(1→3)-galactosyltransferase (GTB) catalyze the final step in ABO(H) blood group A and B antigen synthesis through sugar transfer from activated donor to the H antigen acceptor. These enzymes have a GT-A fold type with characteristic mobile polypeptide loops that cover the active site upon substrate binding and, despite intense investigation, many aspects of substrate specificity and catalysis remain unclear. The structures of GTA, GTB, and their chimeras have been determined to between 1.55 and 1.39 Å resolution in complex with natural donors UDP-Gal, UDP-Glc and, in an attempt to overcome one of the common problems associated with three-dimensional studies, the non-hydrolyzable donor analog UDP-phosphono-galactose (UDP-C-Gal). Whereas the uracil moieties of the donors are observed to maintain a constant location, the sugar moieties lie in four distinct conformations, varying from extended to the "tucked under" conformation associated with catalysis, each stabilized by different hydrogen bonding partners with the enzyme. Further, several structures show clear evidence that the donor sugar is disordered over two of the observed conformations and so provide evidence for stepwise insertion into the active site. Although the natural donors can both assume the tucked under conformation in complex with enzyme, UDP-C-Gal cannot. Whereas UDP-C-Gal was designed to be "isosteric" with natural donor, the small differences in structure imposed by changing the epimeric oxygen atom to carbon appear to render the enzyme incapable of binding the analog in the active conformation and so preclude its use as a substrate mimic in GTA and GTB., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Carbohydrate antigens are valuable as components of vaccines for bacterial infectious agents and human immunodeficiency virus (HIV), and for generating immunotherapeutics against cancer. The crystal structures of anti-carbohydrate antibodies in complex with antigen reveal the key features of antigen recognition and provide information that can guide the design of vaccines, particularly synthetic ones. This review summarizes structural features of anti-carbohydrate antibodies to over 20 antigens, based on six categories of glyco-antigen: (i) the glycan shield of HIV glycoproteins; (ii) tumor epitopes; (iii) glycolipids and blood group A antigen; (iv) internal epitopes of bacterial lipopolysaccharides; (v) terminal epitopes on polysaccharides and oligosaccharides, including a group of antibodies to Kdo-containing Chlamydia epitopes; and (vi) linear homopolysaccharides., (© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Septic shock is a leading cause of death, and it results from an inflammatory cascade triggered by the presence of microbial products in the blood. Certain LPS from Gram-negative bacteria are very potent inducers and are responsible for a high percentage of septic shock cases. Despite decades of research, mAbs specific for lipid A (the endotoxic principle of LPS) have not been successfully developed into a clinical treatment for sepsis. To understand the molecular basis for the observed inability to translate in vitro specificity for lipid A into clinical potential, the structures of antigen-binding fragments of mAbs S1-15 and A6 have been determined both in complex with lipid A carbohydrate backbone and in the unliganded form. The two antibodies have separate germ line origins that generate two markedly different combining-site pockets that are complementary both in shape and charge to the antigen. mAb A6 binds lipid A through both variable light and heavy chain residues, whereas S1-15 utilizes exclusively the variable heavy chain. Both antibodies bind lipid A such that the GlcN-O6 attachment point for the core oligosaccharide is buried in the combining site, which explains the lack of LPS recognition. Longstanding reports of polyspecificity of anti-lipid A antibodies toward single-stranded DNA combined with observed homology of S1-15 and A6 and the reports of several single-stranded DNA-specific mAbs prompted the determination of the structure of S1-15 in complex with single-stranded DNA fragments, which may provide clues about the genesis of autoimmune diseases such as systemic lupus erythematosus, thyroiditis, and rheumatic autoimmune diseases., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The structure of the antigen binding fragment of mAb S25-26, determined to 1.95 Å resolution in complex with the Chlamydiaceae family-specific trisaccharide antigen Kdo(2→8)Kdo(2→4)Kdo (Kdo = 3-deoxy-α-d-manno-oct-2-ulopyranosonic acid), displays a germ-line-coded paratope that differs significantly from previously characterized Chlamydiaceae-specific mAbs despite being raised against the identical immunogen. Unlike the terminal Kdo recognition pocket that promotes cross-reactivity in S25-2-type antibodies, S25-26 and the closely related S25-23 utilize a groove composed of germ-line residues to recognize the entire trisaccharide antigen and so confer strict specificity. Interest in S25-23 was sparked by its rare high μm affinity and strict specificity for the family-specific trisaccharide antigen; however, only the related antibody S25-26 proved amenable to crystallization. The structures of three unliganded forms of S25-26 have a labile complementary-determining region H3 adjacent to significant glycosylation of the variable heavy chain on asparagine 85 in Framework Region 3. Analysis of the glycan reveals a heterogeneous mixture with a common root structure that contains an unusually high number of terminal αGal-Gal moieties. One of the few reported structures of glycosylated mAbs containing these epitopes is the therapeutic antibody Cetuximab; however, unlike Cetuximab, one of the unliganded structures in S25-26 shows significant order in the glycan with appropriate electron density for nine residues. The elucidation of the three-dimensional structure of an αGal-containing N-linked glycan on a mAb variable heavy chain has potential clinical interest, as it has been implicated in allergic response in patients receiving therapeutic antibodies., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)